1. Field of the Invention
The present invention is directed to an application of an inventive systematic strategy for mapping, identification and thereby modification of identified allergenic epitope(s) in proteins with significant reduction or total elimination of human IgE-binding activity for transgenic expression.
2. Description of Prior Art
Plants proteins are the primary source of dietary protein for human and livestock. Most plant proteins, however, are nutritionally incomplete, due mainly to their deficient in certain essential amino acids. Recent advances in plant biotechnology offer new approaches to enhance the protein quality (Sun S. S. M. and Larkin B. A., 1992). Altenbach et al. in 1987 first demonstrated that it was feasible to enhance the methionine content by 30% through transferring and expressing of the heterologous Brazil nut methionine-rich protein (BNMRP) gene in tobacco (Conceicao Ada S. et al., 1994). Subsequently, similar approach and methionine enhancement had been confirmed and shown in Arabidopsis (Altenbach S. B. et al., 1989), rapeseed (Altenbach S. B. et al., 1992; Guerche P. et al., 1990), soybean (Townsend I. A. and Thomas L. A., 1994) and other plants (Aragao F. J. et al., 1992; Saalbach I. et al., 1994; Tu H. M. et al., 1998) without negatively affecting agronomic performance.
The BNMRP, abundant and water-soluble in the seed of Brazil nut (Sun S. S. M. et al., 1987), consists of two low molecular weight polypeptide subunit components, an 8.5-kDa polypeptide and a 3.6-kDa polypeptide, associated through disulfide linkages to form a 12-kDa protein molecule (Sun S. S. M. et al., 1987). The mature protein develops from a larger precursor polypeptide of 18 kDa by multiple stepwise proteolytic cleavages post-translationally (Sun S. S. M. et al., 1987) and is targeted to the protein storage vacuoles in seeds through a protein sorting pathway (Saalbach G et al., 1996). This protein contains 18% methionine as revealed by its amino acid and cDNA sequences (Altenbach S. B. et al., 1987; Ampe C. et al., 1986). This finding triggered the idea of using the BNMRP gene to improve the nutritional quality of some important crops such as legumes. It is well known that the seed protein of legumes is nutritionally incomplete, due to its deficiency in methionine, one of the essential amino acids of human and livestock. By traditional plant breeding approach, improvement of the nutritional quality of legume seeds has not been significant (Payne P. I., 1983), presumably due to the lack of methionine-rich protein genes in legume germplasms.
Unfortunately, during the development of this potential improved product, the BNMRP was identified as a major allergen of the Brazil nut, designated Ber e 1, because it could be recognized by most of the sera from patients allergic to Brazil nut (Arshad S. H. et al., 1991; Asero R. et al., 2002; Bartolome B. et al., 1997; Oommen A. et al., 2000; Pastorello E. A. et al., 1998). It was also demonstrated that a protein extract of transgenic soybeans containing the BNMRP had allergenic activities using radioallergosorbent testing, immunoblotting and skin prick testing (Nordlee J. A. et al., 1996). The potential risk of anaphylaxis hampers the use of native BNMRP for protein quality enhancement in transgenic crops, and further efforts in developing and marketing the methionine-enriched transgenic soybean was halted.
The reason why some particular proteins can cause allergic reactions is not well understood. However, it is well-established that the key step to a specific allergic reaction is the binding of at least two IgE antibody molecules to a multivalent allergenic protein. Binding of the allergen-IgE complex to high affinity IgE receptors on most cells and basophils results in activation of most cells and release of mediators responsible for triggering marked allergic inflammatory responses. Thus, identification and characterization of allergen-specific IgE-binding epitopes known to be either linear or conformational appears to be of crucial importance for the molecular approach to reduce or remove the allergenicity of the BNMRP and a better understanding of the allergenic nature of proteins. Most of the IgE epitopes are supposed to be discontinuous, and are very important for the allergenicity of allergens due to the tertiary structure of proteins. However, our knowledge of structural characteristics of conformational IgE binding sites is very limited (Baerga-Ortiz A., 2002; Bredehorst R. and David K., 2001; Bufe A., 2001; Gonzalez E. M. et al., 2002; Karisola P. et al., 2002; Sen M. et al., 2002). In recent years, through the application of synthetic, overlapping peptides representing the entire primary sequence of a given allergenic protein, multiple distinct linear epitopes have been identified for a variety of allergens including those from cow's milk (Busse P. J. et al., 2002; Chatchatee P. et al., 2001; Chatchatee P. et al., 2001; Jarvinen K. M. et al., 2001), soybean (Helm R. M. et al., 2000; Helm R. M. et al., 2000; Xiang P., 2002), shrimp (Ayuso R. et al., 2002; Reese G et al., 1999; Shanti K. N. et al., 1993), peanut (Burks A. W. et al., 1997; Rabjohn P. et al., 1999; Stanley J. S. et al., 1997; Xiang P. et al., 2002), walnut (Robotham J. M. et al., 2002), pollens (Costa M. A. et al., 2000; Hemmens V. J. et al., 1989; Sakaguchi M. et al., 2001; Schramm G. et al., 2001; Soman K. V. et al., 2000; Suphioglu C. et al., 2001) and other sources (Banerjee B. et al., 2000; Beezhold D. H. et al., 2001; Hemmens V. J. et al., 1989; Kahlert H. et al., 1992; Menendez-Arias L. et al., 1990; Mine Y. and Wei Zhang J., 2002; Monsalve R. I. et al., 1993). There are increasing lines of evidence that linear epitopes play a crucial role as IgE binding sites (Bannon G. A. et al., 2001; Beehold D. H. et al., 2001; Helm R. M. et al., 2000). Much effort had been made to identify epitopes on a variety of allergens including those from foods and other sources, and multiple distinct linear IgE recognition sites were elucidated (Ayuso R. et al., 2002; Banerjee B. et al. 2000; Beezhold D. H. et al., 2001; Burks A. W. et al., 1997; Busse P. J. et al., 2002; Chatchatee P. et al., 2001; Chatchatee P. et al., 2001; Costa M. A. et al., 2000; Helm R. M. et al., 2000; Helm R. M. et al., 2000; Hemmens V. J. et al., 1989; Jarvinen K. M. et al., 2001; Kahlert H. et al., 1992; Menendez-Arias L. et al., 1990; Mine Y. and Wei Zhang J., 2002; Monsalve R. I. et al., 1993; Rabjohn P. et al., 1999; Reese G. et al., 1999; Robotham J. M. et al., 2002; Sakaguchi M. et al., 2001; Schramm G et al., 2001; Shanti K. N. et al., Soman K. V et al., 2000; Stanley J. S. et al., 1997; Suphioglu C. et al., 2001; Xiang P. et al., 2002). The major methods in IgE epitope mapping are based on synthetic, overlapping peptides and recombinant peptide libraries (Reese G. et al., 2001), which are subsequently immunoreacted with human IgE to localize the binding sites. For the BNMRP, the 2S albumin from Brazil nut, however, nothing is known about the structural characteristics of both linear and conformational IgE epitopes so far.